Immunohistochemical markers of small cell carcinoma and related neuroendocrine tumours of the lung

A selected group of 263 pulmonary neuroendocrine tumours comprised 156 small cell carcinomas, five combined cell carcinomas, nine atypical carcinoid/small cell carcinomas, 32 atypical carcinoids, ten large cell/small cell carcinomas, and 51 carcinoid tumours. These were compared with a group of 109 non-small cell carcinomas, using four markers of neuroendocrine differentiation to determine differences in reactivity between the two groups and among the variants of neuroendocrine tumour. The antibodies used were neuron-specific enolase (NSE), protein gene product (PGP) 9.5, human bombesin, and the C-terminal flanking peptide of human bombesin (CTP). Most small cell carcinomas, carcinoid tumours, and atypical carcinoid variants showed immunoreactivity for both NSE and PGP 9.5 but a significant number of non-small cell carcinomas, mainly squamous cell carcinomas, were also positive (11 and 35 per cent, respectively). Bombesin was specific for neuroendocrine tumours, being demonstrable in 35 per cent carcinoids and 24 per cent small cell carcinomas, but staining was focal and often confined to scattered cells. Diffuse strongly positive immunoreactivity for CTP was seen in the majority of malignant neuroendocrine tumours, but only 12 per cent of carcinoid tumours were positive and non-small cell carcinomas were negative. CTP is therefore of potential value as a specific marker of malignant neuroendocrine tumours, particularly if the amount of biopsy material is limited and the tumour is an unusual variant, such as atypical carcinoid or large cell-small cell carcinoma.     

Expression of epithelial and neural antigens in small cell and non small cell lung carcinoma

Seventy-one lung carcinomas from 66 different patients were stained with a panel of monoclonal antibodies. Twenty-nine were small cell lung carcinoma (SCLC), 15 adenocarcinomas, 17 squamous carcinomas and 10 large cell carcinomas. Three of the monoclonal antibodies recognize different cytokeratins, three recognize other epithelial antigens and one recognizes a neural antigen. Both formalin-fixed and cryopreserved tumours were studied using an indirect immunoperoxidase method. 23/29 SCLC reacted with all but one of the antibodies which recognize epithelial antigens. This staining was similar to that seen in non small cell lung carcinomas (NSCLC) and provides further evidence that SCLC are true epithelial tumours. All but one of the SCLC stained with the antibody recognizing a neural antigen. This antibody did not stain squamous or adenocarcinomas. However, four of the large cell carcinomas stained well with this antibody, suggesting that SCLC and some large cell carcinomas share a common pathway of differentiation. There were variations of staining seen both within and between tumours. This has obvious implications if immunotargetting with monoclonal antibodies is to be used diagnostically or therapeutically.     

Expression of the pro-opiomelanocortin gene in lung neuroendocrine tumours: In situ hybridization and immunohistochemical studies

Neuroendocrine tumours of the lung may be associated with the ectopic adrenocorticotrophin (ACTH) syndrome and may synthesize and secrete ACTH-related peptides in the absence of the syndrome. However, immunocytochemical analysis may not confirm these biochemical findings, particularly in small cell carcinoma, which is poorly granulated. To investigate further the morphological evidence for expression of the pro-opiomelanocortin (POMC) gene in neuroendocrine lung tumours, we have examined a series of 46 small cell carcinomas and 13 carcinoid tumours of the lung by in situ hybridization for POMC mRNA using a digoxigenin-labelled oligoprobe. We have compared the findings with the immunocytochemical detection of ACTH and β-endorphin. In situ hybridization was positive in 15 of 46 small cell carcinomas (33 per cent) and in 8 of 13 carcinoid tumours (62 per cent). Immunocytochemical staining was positive in only one carcinoid tumour. These in situ hybridization studies have corroborated biochemical data indicating POMC gene expression in a high proportion of lung neuroendocrine tumours. This suggests that the low levels of expression detected by immunocytochemistry may be due to low levels of hormone storage. Multivariate analysis showed a weak negative association between POMC expression and survival in small cell carcinomas, although this did not reach statistical significance.     

Prealbumin in the diagnosis of bronchopulmonary carcinoid tumours.

The reliability of prealbumin as a diagnostic marker was studied in 60 cases of bronchopulmonary carcinoid tumours. There were differences in the incidence of positivity between typical and atypical carcinoids (well differentiated neuroendocrine carcinomas). Seventy five per cent of the carcinoid tumours were positive for prealbumin; (86.7% typical and 63.3% atypical carcinoids). In 15 cases, which were Grimelius negative, 10 were prealbumin positive. Only 8.3% carcinoids were negative with both prealbumin and Grimelius stains. Ten squamous, 10 adeno- and 10 small cell carcinomas showed only occasional scattered prealbumin positive cells. It is concluded that prealbumin is a useful marker for bronchopulmonary carcinoid tumours. It is cheap, readily available, and should be considered part of routine diagnostic procedures for the diagnosis of carcinoid tumours.     

Detection of endothelin immunoreactivity and mRNA in pulmonary tumours

Paraffin sections of 66 surgically resected lung tumours were immunostained with antisera to human endothelin-1 and to the C-terminal peptide of big endothelin. With both antisera, strong immunoreactivity was demonstrated in 11 of 15 squamous cell carcinomas and 11 of 16 adenocarcinomas. Focal immunoreactivity was seen in small cell carcinoma (2/12), large cell carcinoma (2/5), and carcinoid tumours (2/11). Four lymphomas and three sarcomas did not show endothelin immunoreactivity. Cryostat sections of 22 of the 66 tumours were hybridized with radiolabelled complementary RNA probes prepared from the 3' non-coding region of endothelin-1 cDNA, and the chromosomal genes encoding endothelin-2 and -3. In situ hybridization demonstrated the presence of endothelin mRNAs in 4 of 7 squamous cell carcinomas and in 5 of 8 adenocarcinomas, in a pattern similar to that shown by immunocytochemistry. No hybridization signals were obtained from the other types of tumours. In lung tissue adjacent to the tumours, endothelin-like immunoreactivity and mRNA were detected in pulmonary endocrine cells and, in some cases, other epithelial cells, and in alveolar capillary endothelial cells. This study demonstrates the expression of endothelin in a number of pulmonary tumours and suggests a possible role for this peptide in the growth and/or differentiation of these tumours.     

Distribution and estimation of nucleolar organizer regions in various human lung tumours.

A quantitative study of nucleolar organizer regions in human lung carcinomas was carried out on routinely processed paraffin embedded tissue sections. We examined 104 lung carcinomas including 38 squamous cell carcinomas, 36 adenocarcinomas, 18 large cell anaplastic carcinomas, 6 small cell carcinomas and 6 carcinoids. No significant differences were found in mean number of NORs between squamous, adenocarcinoma and undifferentiated carcinomas including large cell and small cell carcinomas. Carcinoids had comparatively lower means except for one typical carcinoid. Considering the high incidence of overlap between ranges of NOR counts in these groups of tumours and in agreement with the only other study of lung tumours (which comprised only carcinoids and small cell carcinomas), we conclude that this technique cannot be reliably used to discriminate between various histologic types of lung cancers. However, long term follow up of these patients is needed to establish the value of the AgNOR technique for prognostic guidance.     

Expression of the epithelial L1 antigen as an immunohistochemical marker of squamous cell carcinoma of the lung

The distribution of epithelial L1 antigen was evaluated in 139 bronchogenic carcinomas which had been classified by a panel of pathologists according to the WHO recommendation of 1981. L1 was not found in three large cell and 13 small cell carcinomas, but it was expressed by tumour cells in 67 of 69 squamous cell carcinomas (97%), in three of four adenosquamous carcinomas (75%), and in three of 49 adenocarcinomas (6%). The staining for L1 antigen was more diffusely distributed in the positive adenocarcinomas than in the squamous cell carcinomas. Its expression in squamous cell carcinomas was typically confined to relatively small tumour cell groups and never included a complete specimen. Semi-quantitative estimation of the immunostaining showed no clear relationship to the degree of differentiation and scores for proliferation, but L1 expression was negatively related to nuclear aberration (P less than 0.025) and malignancy scores (P less than 0.002). The good agreement between morphological classification and expression of L1 makes this a valuable marker in the diagnosis of lung carcinomas.     

Atypical endocrine tumors of the lung: A histologic, ultrastructural, and clinical study of 19 cases

Lung cancers are divided by light microscopic criteria into several categories, but only two categories are recognized clinically--small cell and non-small cell carcinomas. Transmission electron microscopy has revealed unexpected complexity within each category, blurring the distinctions between them. The present study was undertaken to determine the incidence of dense-core, neuroendocrine-type granules in lung tumors diagnosed by light microscopy as non-small cell carcinomas, i.e., atypical endocrine tumors, and the clinical significance of their identification. Of 205 consecutive primary and metastatic lung cancers, 19 (9 per cent) diagnosed as non-small cell carcinomas by light microscopy were seen to contain neuroendocrine-type granules by electron microscopy and thus were reclassified as atypical endocrine tumors of the lung. Staining with silver stains, periodic acid-Schiff (PAS), PAS with diastase digestion, and mucicarmine was positive in 18, 15, 14, and eight of the 19 cases, respectively. Electron microscopy revealed glandular differentiation in 12 cases and tonofilaments in eight cases, although none of the tumors met the criteria for identification as squamous cell carcinomas. Clinically, the cancers appeared to resemble non-small cell carcinoma more closely than small cell carcinoma. Median survival (12 months) and response to combination chemotherapy (22 per cent) were in the range reported for non-small cell carcinoma. There were no complete responses, despite the use in some cases of regimens active against small cell carcinoma. However, one patient, the only one to date so treated, had a dramatic response to streptozotocin/5-fluorouracil, suggesting that, as in metastatic carcinoid, this combination may have value in the treatment of atypical endocrine tumors of the lung.     

An assessment of involucrin as a diagnostically useful immunohistochemical marker in lung tumours

A previous report has described involucrin as a specific immunohistochemical marker of squamous differentiation in lung carcinomas. The aim of our study was to examine the expression of this antigen in a wide variety of lung tumours, with particular attention to its potential value in the typing of biopsy specimens. We found that immunostaining for involucrin was common in squamous carcinomas but was also found in adenocarcinomas, adenosquamous carcinomas, large cell carcinomas and carcinosarcomas. Small cell carcinomas, carcinoid tumours and mesotheliomas were negative. Contrary to previous claims, this marker appears to have little diagnostic utility in the typing of lung tumours.     

Langerhans cells in human lung tumours: an immunohistological study

In an immunocytochemical study of 41 human lung tumours we have shown that Langerhans cells can be reliably identified using the anti-CD1 monoclonal antibody NA1/34. Langerhans cells are present in all the main varieties of human lung tumour although they are infrequent in both small cell carcinoma and carcinoid tumour. There is considerable variation in numbers of Langerhans cells in both adenocarcinomas and squamous cell carcinomas. In this study tumours were divided into those with high numbers of Langerhans cells (greater than 2 per high power field) and those with low numbers (less than 2 per high power field). Analysing these results against patient survival showed a markedly worse survival in those tumours with a high number of Langerhans cells for all the tumours as a single group and for squamous cell carcinoma as a single entity.     

New approach to assessing lung tumours in man

One hundred and four surgically resected lung tumours were labelled in either cryostat or freeze dried sections with a monoclonal antibody (Ki67), which reacts with a nuclear antigen expressed by proliferating cells. The tumours were categorised semiquantitatively into four proliferative grades, a classification that can be performed rapidly and reproducibly by the pathologist. In keeping with previous cell kinetic studies all small cell carcinomas had high proliferation rates, whereas the carcinoid tumours were in the lowest grade. In contrast, the adenocarcinomas (27 cases) and squamous cell carcinomas (63 cases) varied widely in their proliferative state, in keeping with their heterogeneous, morphological, and clinical behaviour. Immunocytochemical labelling of lung tumour biopsy specimens with antibody Ki67 is a simple technique within the scope of routine surgical pathology laboratories, which might enable these tumours to be classified according to their proliferative status and treatment to be selected accordingly.     

Epithelial markers in primary skin cancer: an immunoperoxidase study of the distribution of epithelial membrane antigen (EMA) and carcinoembryonic antigen (CEA) in 65 primary skin carcinomas

Sixty-five primary malignant skin tumours have been stained for carcinoembryonic antigen (CEA) and epithelial membrane antigen (EMA) using rabbit polyclonal affinity-purified antibodies and an indirect immunoperoxidase technique. The tumours consisted of 15 invasive squamous carcinomas, 23 basal cell carcinomas, 16 malignant eccrine poromas (porocarcinomas), and 11 sebaceous carcinomas. The basal cell carcinomas were negative for CEA and EMA except where there was keratotic or sebaceous differentiation. All the sebaceous and squamous carcinomas and 15/16 porocarcinomas contained EMA. 12/15 squamous carcinomas were positive for CEA. The malignant poromas were negative for CEA except on the ulcerated surface of two. In tumours classified as sebaceous carcinomas there was positive staining for CEA in some cells, cyst contents and/or keratotic foci. These findings have implications for the use of immunoperoxidase localization of epithelial markers in the differential diagnosis of primary and metastatic skin cancer.     

Platelet-derived endothelial cell growth factor (Thymidine Phosphorylase) expression in lung cancer

Platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) is an enzyme with angiogenic and cell motility properties. Moreover, it is involved in the transformation of fluoropyrimidines into active cytotoxic metabolites. In the present study, the expression of PD-ECGF in normal lung and lung cancer was immunohistochemically evaluated using the P-GF.44C monoclonal antibody. Alveolar and tumoural macrophages were invariably stained and were used as an internal control for assessment of the staining. Alveolar epithelium was always negative, whilst bronchiolar epithelium showed occasional positive reactivity. Normal lung and tumour endothelium was occasionally positive. Positive staining in more than 50 per cent of cells was observed in 23/71 squamous carcinomas (32 per cent), 16/38 (42 per cent) adenocarcinomas, and 2/6 (33 per cent) adenosquamous carcinomas. Differentiated areas and areas of squamous metaplasia were more strongly positive than other tumour areas. All 22 small cell carcinomas and one carcinoid tumour were negative. The present study provides a baseline for future studies in non-small cell lung cancer to correlate PD-ECGF expression with tumour vascularization, prognosis, and response to chemotherapy. © 1997 John Wiley & Sons, Ltd.     

Comparison of proliferation rates assessed using "multiblock" and conventional tissue blocks of lung carcinoma.

AIMS: To compare the proliferative rates, assessed immunohistochemically, of human lung tumours using conventional paraffin wax blocks and the multitumour tissue block (MTTB) technique. METHODS: A multiblock containing 20 lung tumours (eight adenocarcinomas, five squamous cell, five small cell and two carcinoid tumours) was constructed. Sections were also cut from the original blocks of formalin fixed, paraffin wax embedded tissue used to construct the multiblock. Sections were stained with the monoclonal antibody PC10, which recognises a proliferating cell nuclear antigen, using the three stage immunoperoxidase technique. RESULTS: The proliferation rates of the lung tumours obtained using both techniques were, overall, significantly different (p = 0.05), although most cases showed good correlation. Some tumours displayed a high degree of intratumoral variation in PC10 staining. The degree of PC10 staining was in keeping with the known proliferative state of particular histological subtypes--that is, carcinoid tumours showed little staining and small cell carcinomas showed extensive positivity. CONCLUSION: The MTTB technique is a less suitable means of assessing proliferation rate in lung carcinomas than conventional tissue blocks. It should be restricted to qualitative antibody studies or quantitative studies using tumours with little intratumoral variation.     

The L1 antigen and squamous metaplasia in the bladder

The L1 antigen was investigated as a marker of squamous differentiation in urothelium using a monoclonal antibody Mac387, and the results were compared with the expression of high molecular weight cytokeratins. L1 antigen was consistently demonstrated in all instances of partial and complete squamous metaplasia and in squamous carcinomas. In contrast, pure transitional cell carcinomas (except one with minor focal staining), adenocarcinomas and normal and hyperplastic urothelium did not label. In a few squamous carcinomas in situ, the pattern of labelling obtained with Mac387 was different from that seen in invasive squamous carcinomas and metaplasias. Compared with high molecular weight cytokeratins, the expression of L1 was more intense in areas of squamous differentiation. L1 expression, as identified by antibody Mac387, may therefore serve as a useful marker of squamous differentiation in urothelial lesions.     

Immunocytochemical localization of neuron specific enolase in small cell carcinomas and carcinoid tumours of the lung

Carcinoid tumours and small cell carcinomas of the lung share many characteristics with normal neuroendocrine cells. While carcinoid tumours contain many dense-cored neurosecretory granules and are frequently argyrophil, small cell carcinomas are poorly granulated and rarely argyrophil, which casts doubt on their neuroendocrine nature. Immunostaining of the enzyme neuron specific enolase (NSE) was recently used to demonstrate the neuroendocrine components of the lung including nerves and neuroendocrine cells. We therefore used NSE immunostaining to investigate neuroendocrine differentiation in 79 lung tumours, including 18 bronchial carcinoids and 31 small cell carcinomas, and compared these results with those obtained with silver stains. Thirteen of the 18 carcinoids were reactive to silver, all other types being negative. NSE-immunoreactivity occurred in 16 carcinoids and 18 small cell carcinomas. None of the squamous cell carcinomas, large cell anaplastic carcinomas and adenocarcinomas examined showed NSE-immunoreactivity. Radioimmunoassay of extractable NSE from 10 fresh lung tumours correlated well with the immunostaining results, demonstrating large amounts in two small cell carcinomas (334 and 517 ng/mg protein) and three carcinoids (152, 908, and 1143 ng/mg protein). Values were much lower for four squamous cell carcinomas (31-44 ng/mg protein) and one large cell anaplastic carcinoma (30 ng/mg protein) and were accounted for by the presence of NSE-positive nerves and neuroendocrine cells in the surrounding lung. NSE immunostaining is a useful marker of neuroendocrine differentiation in lung tumours and should prove particularly valuable in the diagnosis of small cell anaplastic tumours and their metastases.     

Squamous differentiation in primary urothelial carcinoma of the urinary tract as seen by MAC387 immunohistochemistry

Squamous differentiation (SqD) is variably present in urinary tract tumours, but its significance remains unclear. In this study, SqD was assessed by immunohistochemistry using the monoclonal antibody Mac387 in 145 urothelial tumours (bladder, n = 115; renal pelvis, n = 30). Mac387 detects the myelomonocytic L1 antigen; a member of the calgranulin family shared by epithelial cells and keratinocytes. L1 antigen was shown in SqD in urothelial carcinomas of the bladder or the renal pelvis, including 11 cases with focal SqD unrecognised by conventional analysis. SqD is more frequent in renal pelvic tumours (p = 0.027) and increases with grade/stage mainly in bladder carcinoma (grade, p = 0.05; stage, p = 0.005). Stage Ta/T1 bladder carcinomas with SqD recurred more (p = 0.021). In conclusion, Mac387 efficiently shows SqD in urothelial tumours.     

Are keratin proteins a better tumor marker than epithelial membrane antigen? A comparative immunohistochemical study of various paraffin-embedded neoplasms using monoclonal and polyclonal antibodies

Epithelial membrane antigen and keratin proteins represent markers of epithelial differentiation that may be detected in routine formalin-fixed, paraffin-embedded tissues. Eighty-seven neoplasms, including 48 adenocarcinomas of various types, squamous and transitional cell carcinomas, small-cell anaplastic carcinomas, carcinoid tumors, mesotheliomas, hepatomas, melanomas (metastatic), adrenal cortical carcinomas, germ cell tumors, and extramammary Paget's disease, were assessed to determine the relative effectiveness of these antigens as tumor markers. Immunoperoxidase studies were performed using monoclonal antibodies to epithelial membrane antigen and monoclonal (combined AE1 and AE3) and polyclonal (bovine muzzle keratins) antibodies to keratin proteins. In more than half the cases (50/87%), both markers yielded comparable results. However, in 29 cases (33%), keratin proteins were clearly superior to epithelial membrane antigen as a tumor cell marker. Particular discrepancies were apparent for some gastrointestinal adenocarcinomas, squamous cell carcinomas, hepatomas (hepatocellular type), spindle cell components of mesotheliomas, and carcinoid tumors. Epithelial membrane antigen represented a better marker in eight cases (9%), mainly for small-cell anaplastic carcinomas and some renal cell and pulmonary adenocarcinomas. Adrenal cortical carcinomas, melanomas, and seminomas were nonimmunoreactive for both antigens. Epithelial membrane antigen and keratin proteins represent useful complementary markers in diagnostic surgical pathology.     

Differential expression of neural-cadherin in pulmonary epithelial tumours

Aims:  Neural (N)-cadherin belongs to a group of transmembrane molecules with a crucial role in tissue morphogenesis and maintenance of an epithelioid phenotype and increased N-cadherin expression is implicated in tumour progression and dedifferentiation. The aim was to determine whether evaluation of N-cadherin in pulmonary tumours might assist in identifying lesions with more aggressive potential.
Methods and results:  One hundred and fifty-five pulmonary lesions were analysed for N-cadherin expression using immunohistochemistry, including neuroendocrine hyperplasia ( n  = 3), typical carcinoid ( n  = 59), atypical carcinoid ( n  = 12), small cell lung carcinoma ( n  = 11), large cell neuroendocrine carcinoma ( n  = 12), adenocarcinoma ( n  = 35) and squamous cell carcinoma ( n  = 23). Lymph node status was correlated with immunohistochemical expression. N-cadherin expression was demonstrated in all cases of neuroendocrine hyperplasia, 96% of typical carcinoids, 83% of atypical carcinoids, 63% of the small cell lung carcinomas and 32% of large cell neuroendocrine carcinomas. Over 90% of the adenocarcinomas and 100% of the squamous cell carcinomas were negative. Increased N-cadherin expression in typical carcinoids was associated with negative lymph node status ( P  < 0.001).
Discussion:  N-cadherin is differentially expressed in pulmonary tumours and is predominantly observed in neuroendocrine lung lesions with high expression in typical and atypical pulmonary carcinoids. The level of expression of N-cadherin between types of lung tumours does not appear to indicate malignant potential or aggressive behaviour.